The long term objective of this research is to develop an understanding of the factors that govern RNA folding. Because of the connection between structure and biological activity, defining these factors should lead to a better understanding of how ribonucleic acids perform their functions in vivo. In practical terms, this knowledge will facilitate efforts both to predict the three-dimensional structure of complex RNAs from primary sequence, and to design RNA molecules that adopt stable structures possessing specific binding and catalytic properties. Such RNAs hold considerable promise as biochemical tools, diagnostic reagents, and as starting points for the development of therapeutics for human disease. A central aspect of the proposed research for the next funding cycle will be the application of modified RNA bases to probe kinetic, thermodynamic, and structural aspects of tertiary folding transitions. This a critical, but relatively unexplored aspect of RNA folding. In the first specific aim, the role that conserved hydrogen bonds play in tertiary folding will be determined by synthesizing conservative nondisruptive deletion mutants that lack specific donor and/or acceptor groups. Each mutation is designed to act as a reporter for folding in a particular region of tRNA tertiary structure. Rates and equilibrium constants will be measured for each mutant to obtain activation and equilibrium free energies of folding, respectively. These data will be used to construct difference free energy diagrams that will illustrate the role of various interactions in the course of tertiary folding. In the second aim, a set of tRNAs each containing single pair of thiols will be synthesized, and the effective concentration (Ceff) of each thiol pair will be measured at a series of fixed [Mg2+] to monitor the formation of tRNAPhe tertiary structure at equilibrium. As part of this work, the thermodynamic linkage between disulfide bond formation and tRNA stability will be exploited to quantify the effects of point mutations on tRNA stability. These experiments represent a new and powerful method to investigate nucleic acid thermodynamics which has the capacity to determine if changes in primary sequence affect secondary structure, tertiary structure or both of these. In the final aim of this grant renewal, NMR spectroscopy will be used to determine the [Mg2+] when the tertiary interactions in tRNAPhe form. These experiments are designed to interpret the effective concentration data obtained in Aim 2 in a structural context.